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J. Mar. Sci. Eng. 2017, 5(1), 4; doi:10.3390/jmse5010004

Evaluating the Reliability of Counting Bacteria Using Epifluorescence Microscopy

1
Department of Marine Science and Fisheries, College of Agricultural and Marine Sciences, Sultan Qaboos University, PO Box 34, Al Khoud, Muscat 123, Sultanate of Oman
2
Center of Excellence in Marine Biotechnology, Sultan Qaboos University, PO Box 50, Al Khoud, Muscat 123, Sultanate of Oman
3
Department of Biology, College of Science, Sultan Qaboos University, PO Box 36, Al Khoud, Muscat 123, Sultanate of Oman
*
Author to whom correspondence should be addressed.
Academic Editor: Magnus Wahlberg
Received: 25 October 2016 / Revised: 6 December 2016 / Accepted: 8 December 2016 / Published: 8 January 2017
(This article belongs to the Section Marine Biology)
View Full-Text   |   Download PDF [3475 KB, uploaded 9 January 2017]   |  

Abstract

The common practice of counting bacteria using epifluorescence microscopy involves selecting 5–30 random fields of view on a glass slide to calculate the arithmetic mean which is then used to estimate the total bacterial abundance. However, not much is known about the accuracy of the arithmetic mean when it is calculated by selecting random fields of view and its effect on the overall abundance. The aim of this study is to evaluate the accuracy and reliability of the arithmetic mean by estimating total bacterial abundance and to calculate its variance using a bootstrapping technique. Three fixed suspensions obtained from a three-week-old marine biofilm were stained and dispersed on glass slides. Bacterial cells were counted from a total of 13,924 fields of view on each slide. Total bacterial count data obtained were used for calculating the arithmetic mean and associated variance and bias for sample field sizes of 5, 10, 15, 20, 25, 30, 35 and 40. The study revealed a non-uniform distribution of bacterial cells on the glass slide. A minimum of 20 random fields of view or a minimum of 350 bacterial cells need to be counted to obtain a reliable value of the arithmetic mean to estimate the total bacterial abundance for a marine biofilm sample dispersed on a glass slide. View Full-Text
Keywords: epifluorescence; bacteria; count; random; arithmetic mean; bootstrapping; normal distribution epifluorescence; bacteria; count; random; arithmetic mean; bootstrapping; normal distribution
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Muthukrishnan, T.; Govender, A.; Dobretsov, S.; Abed, R.M. Evaluating the Reliability of Counting Bacteria Using Epifluorescence Microscopy. J. Mar. Sci. Eng. 2017, 5, 4.

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